Bacterial lipopolysaccharides (LPSs) are characteristic outer membrane constituents of Gram-negative bacteria. LPSs are widely used as antigens in diagnostic assays specially designed for the specific detection of antibodies in serum, plasma, meat juice, saliva or other body fluids, originating from bacterial infections in humans and animals. LPSs are highly immunogenic and comprise one of the epitope characteristics for a given bacterial strain. In fact, the definition of a serotype is often based on the LPS and/or capsular polysaccharide (CPS) antigenicity. The antigenic specificity of the LPS molecule resides in the polysaccharide part of the LPS, the O-antigen, whereas the toxicity of the LPS is caused by residues contained in the lipid part of the LPS, called the lipid A. LPSs are highly amphiphilic compounds because of the joint presence of a hydrophilic O-polysaccharide group and a hydrophobic lipid group in the LPS molecule. Most of the characterised LPSs have the same principal structure which is especially conserved in the lipid A and in the inner core parts of the LPSs. The core is the part of the polysaccharide that comprises the bond between the O-antigen and the lipid A. This bond is invariably comprised of a ketosidic bond between the hemiketal function of the innermost KDO-residue and a hydroxyl-group of a GIcNAc-residue of the lipid A. The O-antigen of a specific bacterial serotype varies with respect to numbers of repeating units and may contain non-stoicheometrical substitutions with acetyl, phosphate, glycosyl or other groups. Generally, LPS-molecules without O-antigens, that is carrying only (parts of the core saccharides in addition to the lipid A are called “rough” LPS, while LPS-molecules carrying O-antigens are called “smooth” (Raetz, C. R. H. in Escherichia coli and Salmonella: Cellular and Molecular Biology (Neidhardt, F. C. E. A., ed.) Vol. 1, 2nd Ed., pp. 1035-1063, American Society for Microbiology, Washington D.C., 1996; Hitchcock et al, 1986, J. Bacteriol. 166, 699-705).
ELISA, enzyme-linked immunosorbent assay, is a well known method for detection of antibodies. In this assay, the LPSs are coated (or immobilised) on a solid surface (e.g. a plastic surface) by passive adsorption, where they serve as probes for specific antibodies. The method consists of incubation of the LPS-coated surface with the biological sample being assayed for the presence of antibodies, followed by incubation of the LPS-antibody complex with a labelled secondary antibody.
Previously, LPSs have generally been immobilised onto a solid surface without any modification of the molecules since the hydrophobic lipid A part of the molecules functions as a fairly efficient “anchor” binding the LPSs to the surface via non-covalent hydrophobic interactions leaving the hydrophilic O-polysaccharides pointing outward accessible for interactions with binding components, e.g. antibodies. However, it has been shown that the efficiency by which the LPSs are immobilised onto hydrophobic surfaces depends on both the nature of the surface and the equilibrium between free LPSs and formed LPS micelles. The equilibrium between free LPSs and formed LPS micelles depends on the amphiphilic nature of the LPSs and varies between LPSs from different bacteria strains as well as between different LPS serotypes. Certain types of LPSs have shown to be very difficult to immobilise onto solid surfaces by non-covalent bonds without addition of various micelle-dispersing agents (detergents) to the coating solution.
Thus, the optimal coating conditions vary among LPS from different bacteria strains as well as between serotypes of the same bacteria, making simultaneous immobilisation of two or more different LPSs onto the same surface very difficult. This is envisaged to be due to the ability of a well-coating LPS type to compete out the less well-coating type. This phenomenon was illustrated with Salmonella Infantis LPS which was shown to coat inefficiently to plastic. In an assay for detection of Salmonella Typhimurium nd Salmonella Infantis specific antibodies this lead to the substitution of Salmonella Infantis LPS with Salmonella Choleraesuis LPS which was found to coat much better and which carries the same antigenicserotypes (O-antigens) (Nielsen, B. et al., 1995, Vet. Microbiol. 47, 205-218)
The tendency of LPS to form micelles (or aggregates) is furthermore believed to reduce the stability of the LPS coating as the interactions between bimolecular (or aggregated or cluster) LPS and the surface are believed to be weaker then the interactions between a single molecule of LPS and the surface. In other words, the potential tendency of LPS to coat in clusters may lead to a decreased and unpredictable coating stability and reduce the long-term stability of the coating.
EP 0 101 119 describes the immobilisation of a lipopolysaccharide to an insoluble carrier by the condensation of the lipopolysaccharide and either an amino or carboxyl group of the insoluble carrier.
JP-A-2-242448 (Patent Abstract of Japan) describes the immobilisation of a lipid A glycoside of 3-deoxy-D-manno-2-octurosonic acid to the surface of an insoluble carrier through an amide bond.
Highly hydrophilic antigens like e.g. bacterial polysaccharide (PS) are often very difficult to adsorb (immobilise) onto the most commonly used surfaces used in serological assays, such as plastics used in ELISA and RIA (radio-immuno assay), latex particles used in agglutination techniques and PVDF (polyvinylidenedifluoride) as well as nitrocellulose and other materials used for dip-stick, blotting or other fast assays. Accordingly, PS as such coat inefficiently and demand the use of large quantities of polysaccharide antigen or extremes of pH and can not be used with mixtures of polysaccharides (Elkins et al., 1990, J. Immunol. Meth. 130,123-131). On the contrary, in spite of the drawbacks mentioned above LPSs are almost exclusively used as coating antigens as the lipid A provides the required hydrophobicity needed for adequate coating of the surface with antigen.
PSs have previously been isolated from LPSs with the purpose of preparing conjugates of PS and carrier substances, most often carrier proteins for vaccine purposes (Aron et al., 1993, J. Clin. Microbiol. 31, 975-978; Lambden and Heckels, 1982, J. Immunol. Meth. 48, 233-240; Gupta et al., 1995, Inf. Immun. 63, 2805-2810).
Previously, it has been shown that the polysaccharide part of Salmonella Typhimurium lipopolysaccharide (LPS) could be derivatised with biotin and immobilised onto a streptavidin-coated ELISA-plate where it could be recognised by antibodies against PS.
By using the hydrazide derivative of biotin, it was possible to react the PS directly with biotin-hydrazide without a prior oxidation step; the hydrazide was shown to react with the hemiketal of the reducing end KDO of the PS. This procedure while leading to antigenically intact biotinylated PS-derivatives, however resulted in derivatives that were not stable, and an avidin or streptavidin coating had to be introduced in the assay (Wiuff, C., Lind, P., Heegaard, P., 1997, Regioselective coupling of reducing carbohydrates to hydrazides for derivatization of bacterial polysaccharides and application to immunoassays, Proc. 2nd. Carbohydrate Engineering Meeting, La Rochelle, France, p. 66).
In Meikle et al. (Glycoconjugate J. 7, 207-218,1990) a PS is used in an ELISA by directly coupling the PS to a detection enzyme in a competitive set-up. The coupling was performed by reductive amination of the keto functionality of the keto-carboxy group of a KDO unit of the PS. The PS/detection enzyme conjugate was not used for coating but was used in solution in a later step in the assay.
Generally, binding of PS to solid surfaces requires modifications of the PS molecule, but in such a way the O-antigen part stays unaltered. A number of such modifications have been described for naturally occurring polysaccharides. A well-known example is the coupling of capsular polysaccharides (CPSs), which do not contain hydrophobic parts (or groups), to proteins (Laferriere et al., 1997, Vaccine 15,179-186; Beuvery et al., 1986, Develop. biol. Standard. 63,117-128.). The resulting capsular polysaccharide-protein complexes are then adsorbed to the surfaces through hydrophobic groups in the carrier protein. Bacterial capsular polysaccharides have also been modified by non-regioselective reaction of hydroxyl groups with hydrophobic groups e.g. phenyl or thyramine to enhance the overall hydrophobicity of the CPS molecules and there by the binding abilities to solid surfaces (Kristensen and Bentzon, 1992, APMIS 100, 142-146).
A major problem is that antigenicity may be difficult to retain after derivatisation of the polysaccharide, as most methods are not directed to the derivatisation of any specific region of the polysaccharide antigen, thereby possibly destroying or modifying antigenic epitopes of the polysaccharide.
This has been overcome with the present invention by restricting the derivatisation to certain regions of the carbohydrate.